Telomerase prevents the shortening of the ends of each chromosome that normally occurs with every cell division. Telomerase is repressed in most normal cells, and serves as a brake against the progression of premalignant cells by limiting the number of divisions they can undergo while accumulating mutations. Almost all cancers have had to upregulate/reactivate telomerase in order to overcome this limit. Telomerase is regarded as an ideal cancer therapeutic target because of its required expression in ~85% of all tumors and its absence in most normal cells. However, progress towards identifying good inhibitors has been slow and only one, an oligonucleotide directed at the catalytic site of the telomerase template RNA, is in Phase I/II clinical trials. This application proposes to investigate alternative splicing of the telomerase mRNA as a potential additional target for increasing the effectiveness of telomerase inhibition. Less than 5% of the total telomerase (hTERT) mRNA is spliced to give a full length mRNA capable of being translated into active protein. Three approaches for manipulating this splicing to reduce the amount of functional protein produced will be used: 1) Oligonucleotides directed against splicing factor binding sites have been shown to be able to cause exon-skipping, and are in clinical trials for a number of genetic diseases. We have already identified a variety of oligonucleotides that can increase non-functional alternative spliced telomerase mRNAs. These and others will be characterized and refined in order to optimize their effectiveness; 2) We have created a minigene that recapitulates the important alternatively spliced variants of telomerase. A deletion analysis will be used to define the important regulatory sequences that control telomerase alternative splicing. Using that knowledge, the important RNA sequences will be used to affinity purify and identify splicing factors important for telomerase alternative splicing; and 3) The minigene characterized above will be used to create a screen that will allow the preferential survival of cells with decreased full-length mRNA. We will infect shRNA or cDNA libraries to identify splicing factors important for the regulation of telomerase splicing. The knowledge and reagents created using the approaches above will permit chemical screens to be developed in order to identify small molecules capable of reducing full-length telomerase splice forms by increasing the fraction of non-functional alternatively spliced messages. Telomerase inhibition has the potential to be an almost universal treatment for cancer. This project will expand the molecular targets capable of inhibiting telomerase and may finally lead to the development of highly effective telomerase inhibitors.